Post-transcriptional modifications are common in RNA molecules to expand their nucleotide repertoire, but due to limited functional hypotheses and analytical techniques many RNA modifications remain uncharacterized. N6-methyladenosine (m6A) is the most common internal base modification on eukaryotic messenger RNA (mRNA). To date nothing is known regarding the function of this modification. To elucidate this function, our research focused on three approaches: first, mapping the positions of m6A in S. cerevisiae, D. melanogaster, and zebrafish by combining immunoprecipitation using an antibody that binds specifically to m6A with high-throughput sequencing. Second, using in-vitro transcription and biotinylated, methylated RNA fragments to pull down proteins specifically associated with m6A. Third, designing constructs that harbor an endogenous methylation consensus site, as well as counterparts in which a single point mutation eliminated the consensus site. Such constructs offer the potential of studying the role of m6A in the life cycle of RNA. Our results provided insight into genome-wide trends of this modification, suggesting that zebrafish show similar conservation to the human and mouse genomes. In addition, we have pulled down potential protein candidates that will be identified in the future using mass spectrometry. By exploring the m6A landscape in model organisms, the proteins binding to this modification and its effect in RNA’s life cycle, we hope to understand the functions of this common, highly-conserved modification.
PROJECT: Mapping and Exploring the Functions of N6-methyladenosine in mRNA
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